1. Development of hydroxylated naphthylchalcones as polyphenol oxidase
inhibitors: Synthesis, biochemistry and molecular docking studies
Sini Radhakrishnan ⇑
, Ronald Shimmon, Costa Conn, Anthony Baker
School of Chemistry and Forensic Science, University of Technology Sydney, 15 Broadway, Ultimo, NSW 2007, Australia
a r t i c l e i n f o
Article history:
Received 8 September 2015
Revised 7 October 2015
Accepted 8 October 2015
Keywords:
Tyrosinase
Competitive
Docking
Reversible
a b s t r a c t
Polyphenol oxidase (Tyrosinase) has received great attention, since it is the key enzyme in melanin
biosynthesis. In this study, novel hydroxy naphthylchalcone compounds were synthesized, and their
inhibitory effects on mushroom tyrosinase activity were evaluated. The structures of the compounds
synthesized were confirmed by 1
H NMR, 13
C NMR, FTIR and HRMS. Two of the compounds synthesized
inhibited the diphenolase activity of tyrosinase in a dose dependent manner and exhibited much higher
tyrosinase inhibitory activities (IC50 values of 10.4 lM and 14.4 lM, respectively) than the positive con-
trol, kojic acid (IC50: 27.5 lM). Kinetic analysis showed that their inhibition was reversible. Both the novel
compounds displayed competitive inhibition with their Ki values of 3.8 lM and 4.5 lM, respectively.
Docking results confirmed that the active inhibitors strongly interacted with the mushroom tyrosinase
residues. This study suggests hydroxy naphthylchalcone compounds to serve as promising candidates
for use as depigmentation agents.
Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction
Polyphenol oxidase (PPO), also known as tyrosinase [EC
1.14.18.1] is a multifunctional copper-containing enzyme, widely
distributed in micro-organisms, animals, and plants [3,15]. The
major rate limiting step in melanin biosynthesis involves the
enzyme polyphenol oxidase that catalyzes two different reactions
of melanin biosynthesis, the hydroxylation of L-tyrosine to
L-DOPA (L-3,4-dihydroxy phenylalanine) and oxidation of L-DOPA
to DOPA quinone [16]. From a structural perspective, tyrosinase
has two copper ions in its active site which play a vital role in its
catalytic activity. At the active site of tyrosinase, a dioxygen
molecule binds in side-on coordination mode between two copper
ions. Each of the copper ions is coordinated by three histidines in
the protein matrix [19]. The copper atoms participate directly in
hydroxylation of monophenols to diphenols (cresolase activity)
and in the oxidation of o-diphenols to o-quinones (catechol oxidase
activity) that enhance the production of the brown color [4,10].
Therefore, chelation of tyrosinase Cu2+
by synthetic compounds
or agents from natural sources has been targeted as a way to
inhibit or block tyrosinase catalysis [21]. An alternative solution
to inhibit tyrosinase catalytic activity would be by effectively
blocking access to the active site of enzyme.
Alterations in tyrosinase dysfunction could culminate with
serious pigmentation disorders like melasma, chloasma, lentigo,
age spots, inflammatory hypermelanosis and trauma-induced
hyperpigmentation [17,1,26]. Thus use of tyrosinase inhibitors is
becoming increasingly important in the cosmetic and medicinal
industries due to their preventive effect on pigmentation disorders.
In addition, tyrosinase is responsible for undesired enzymatic
browning of fruits and vegetables that take place during
senescence or damage in post-harvest handling, which makes the
identification of novel tyrosinase inhibitors extremely important.
Enzymatic browning could culminate with discoloration and a
decline in the nutritional value of foods [18]. Recently, 4-hexyl
resorcinol has found to be quite effective for browning control in
fresh and dried fruit slices [20]. Despite reports of a large number
of tyrosinase inhibitors, only a few are used today because of their
limitations with regard to cytotoxicity, selectivity, and stability
[12,6,25,5].
Studies have shown hydroxy-substituted chalcones to have
good tyrosinase inhibition [14,11]. Previously, we have reported
the synthesis of hydroxy substituted azachalcone compounds with
potential inhibitory effects on mushroom tyrosinase activity [22].
Furthermore, several hydroxy-substituted 2-phenylnaphthalene
derivatives were reported as potent tyrosinase inhibitors. Among
them, 4-(6-hydroxy-2-naphthyl)-1,3-benzendiol (HNB) and 5-(6-
hydroxy-2-naphthyl)-1,2,3-benzentriol (5HNB) were found to
inhibit mushroom tyrosinase activity [24,9,2]. Hence, in our
http://dx.doi.org/10.1016/j.bioorg.2015.10.003
0045-2068/Ó 2015 Elsevier Inc. All rights reserved.
⇑ Corresponding author.
E-mail address: sinikaranayil@gmail.com (S. Radhakrishnan).
Bioorganic Chemistry 63 (2015) 116–122
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg

2. continuous effort to search for potential tyrosinase inhibitors and
to develop a new template, we have attempted to design and
synthesize a series of novel hydroxy substituted naphthylchalcone
compounds for application as effective polyphenol oxidase
inhibitors. We hypothesized that the ligand’s hydroxyl group
may block polyphenol oxidase activity by binding to the copper
atoms in the active site of mushroom tyrosinase based on the fact
that previous findings had shown the role of hydroxyl groups in
tyrosinase inhibition [7,13]. Assays were performed with L-DOPA
as the substrate, using kojic acid, a well-known strong tyrosinase
inhibitor as the positive control. We have further investigated
the kinetic parameters and inhibition mechanisms of active tyrosi-
nase inhibitor compounds. In addition, we determined the IC50,
that is, the concentration of compound that causes 50% inhibition
for kojic acid and the active inhibitors. Dose-dependent inhibition
experiments were performed in triplicate to determine the IC50 of
test compounds. To confirm our hypothesis, we simulated the
docking between the ligands and tyrosinase using Discovery
Studio 4.5. From the docking results, we checked for possible
hydrogen-bonding and non-bonding interactions with the amino
acid residues. For the control simulation, the docking simulation
of kojic acid, a well-known tyrosinase inhibitor, with tyrosinase
was also performed.
2. Materials and methods
2.1. Chemical reagents and instruments
Melting points (Mp) were determined with WRS-1B melting
point apparatus and the thermometer was uncorrected. NMR
spectra were recorded on Agilent 500 spectrometer at 25 °C in
CDCl3 or DMSO-d6. All chemical shifts (o) are quoted in parts per
million downfield from TMS and coupling constants (J) are given
in Hz. Abbreviations used in the splitting pattern were as follows:
s = singlet, d = doublet, t = triplet, m = multiplet. HRMS spectra
were recorded using the Agilent Technologies 6520 LC/MS-QTOF.
All reactions were monitored by TLC (Merck Kieselgel 60 F254)
and the spots were visualized under UV light. Infrared (IR) spectra
were recorded on Thermo Scientific NICOLET 6700 FT-IR spectrom-
eter. Tyrosinase, L-3,4-dihydroxyphenylalanine (L-DOPA) and kojic
acid were purchased from Sigma–Aldrich Chemical Co.
2.2. General method for the synthesis of naphthylchalcones (1a–5a)
To a stirred solution of 20
-hydroxy-10
-acetonaphthone (1 mM)
and a substituted aldehyde (1 mM) in 25 ml methanol, was added
pulverized NaOH (2 mM) and the mixture was stirred at room
temperature for 24–36 h. The reaction was monitored by TLC using
n-hexane: ethyl acetate (7:3) as mobile phase. The reaction
mixture was cooled to 0 °C (ice-water bath) and acidified with
HCl (10% v/v aqueous solution) to afford total precipitation of the
compounds. In most cases, a yellow precipitate was formed, which
was filtered and washed with 10% aqueous HCl solution. In the
cases where an orange oil was formed, the mixture was extracted
with CH2Cl2, the extracts were dried (Na2SO4) and the solvent was
evaporated to give the respective chalcone (1a–5a).
(1a). (2E)-3-(2,5-dimethoxyphenyl)-1-(3-hydroxynaphthalen-2
-yl) prop-2-en-1-one. M.p 114–116 °C. 1
H NMR (500 MHz, CDCl3):
o 12.54 (s, 1H, OH), 8.12 (dd, 1H, J = 12.5, H-100
), 7.96 (d, 1H,
J = 13.5, Ha), 7.85 (s, 1H, H-6), 7.80 (dd, 1H, J = 10.0, H-50
), 7.67
(dd, 1H, J = 8.5, H-70
), 7.59 (dd, 1H, J = 9.0, H-80
), 7.52 (dd, 1H,
J = 9.5, H-60
), 7.49 (d, 1H, J = 9.5, H-30
), 6.92 (d, 1H, J = 13.5, Hb),
6.57 (dd, 1H, J = 10.0, H-4), 6.49 (dd, 1H, J = 9.5, H-3), 3.82 (s,
3H), 3.80 (s, 3H); 13
C NMR (125 MHz, DMSO-d6) o 195.2 (C@O),
116.5 (C10
), 166.2 (C20
), 123.2 (C30
), 136.1 (C40
), 130.4 (C50
), 134.0
(C60
), 128.4 (C70
), 130.0 (C80
), 122.4 (C90
), 131.2 (C100
), 142.4
(vinylic), 131.7 (vinylic), 129.2 (C1), 156.6 (C2), 112.3 (C3), 126.4
(C4), 153.2 (C5), 112.2 (C6), 56.2 (Me); IR (KBr) m (cmÀ1
): 3022,
2992, 2773, 1682, 1550, 1425, 1046, 814, 707; MS (ESI): 335.1
([M+H])+
.
(2a). (2E)-3-(2,4-dimethoxyphenyl)-1-(3-hydroxynaphthalen-
2-yl) prop-2-en-1-one. M.p 87–89 °C. 1
H NMR (500 MHz, CDCl3):
o 12.72 (s, 1H, OH), 8.19 (dd, 1H, J = 15.0, H-100
), 7.98 (d, 1H,
J = 15.0, Ha), 7.82 (dd, 1H, J = 12.0, H-50
), 7.78 (dd, 1H, J = 10.0,
H-6), 7.63 (t, 1H, J = 8.5, H-70
), 7.57 (dd, 1H, J = 8.0, H-80
), 7.49
(dd, 1H, J = 9.0, H-60
), 7.47 (d, 1H, J = 9.0, H-30
), 7.09 (d, 1H,
J = 15.0, Hb), 6.53 (t, 1H, J = 16.0, H-5), 6.44 (m, 1H, J = 9.0, H-3),
3.86 (s, 3H), 3.88 (s, 3H); 13
C NMR (125 MHz, DMSO-d6) o 194.6
(C@O), 114.1 (C10
), 165.0 (C20
), 122.2 (C30
), 137.1 (C40
), 130.4
(C50
), 132.6 (C60
), 126.1 (C70
), 128.4 (C80
), 124.4 (C90
), 133.2
(C100
), 140.8 (vinylic), 130.5 (vinylic), 119.2 (C1), 162.6 (C2), 99.3
(C3), 166.2 (C4), 107.2 (C5), 130.2 (C6), 55.6 (Me); IR (KBr) m
(cmÀ1
): 3018, 2957, 2781, 2614, 1679, 1562, 1427, 1375, 1218,
1102, 817, 717, 605; MS (ESI): 335.1 ([M+H])+
.
(3a). (2E)-1-(2-hydroxyphenyl)-3-(2-methoxy-4-nitrophenyl)
prop-2-en-1-one Mp: 123–125 °C. 1
H NMR (500 MHz, CDCl3): o
12.22 (s, 1H, OH), 8.22 (s, 1H), 7.95 (d, 1H, J = 14.0, Hb), 7.86 (m,
1H, J = 9.5, H-6), 7.80 (d, IH, J = 14.0, Ha), 7.75 (d, 1H, J = 10.5,
H-60
), 7.69 (d, 1H, J = 8.5, H-5), 7.59 (dd, 1H, J = 8.5, H-40
), 7.02
(dd, 1H, J = 9.0, H-30
), 6.97 (m, 1H, J = 10.0, H-50
), 3.82 (s, 3H);
13
C NMR (125 MHz, DMSO-d6) o 194.2 (C@O), 130.2 (C60
), 119.3
(C10
), 118.2 (C50
), 137.0 (C40
), 118.4 (C30
), 162.9 (C20
), 128.2 (C1),
115.2 (C2), 112.2 (C3), 137.5 (C4), 122.4 (C5), 116.5 (C6), 125.5
(vinylic), 140.8 (vinylic); IR (KBr) m (cmÀ1
): 3250, 3345, 3015,
2910, 2865, 2700, 1702, 1682, 1552, 1465, 1305, 979, 735, 680,
575; MS (ESI): 270.1 ([M+H])+
.
4a. (2E)-1-(2-hydroxyphenyl)-3-(4-methoxy-3-nitrophenyl)
prop-2-en-1-one Mp: 110–112 °C. 1
H NMR (500 MHz, CDCl3): o
12.47 (s, 1H, OH), 8.10 (s, 1H), 7.86 (d, 1H, J = 12.5, Hb), 7.74 (d,
1H, J = 10.0, H-60
), 7.72 (d, IH, J = 12.5, Ha), 7.65 (d, 1H, J = 9.5,
H-5), 7.54 (d, 1H, J = 8.0, H-6), 7.49 (t, 1H, J = 8.5, H-40
), 6.92 (dd,
1H, J = 9.5, H-30
), 6.90 (dd, 1H, J = 9.0, H-50
), 3.86 (s, 3H); 13
C NMR
(125 MHz, DMSO-d6) o 192.22(C@O), 130.52 (C60
), 119.25 (C10
),
118.27 (C50
), 136.77 (C40
), 118.28 (C30
), 161.92 (C20
), 126.90 (C1),
120.54(C2), 137.22 (C3), 147.50 (C4), 112.42 (C5), 114.23 (C6),
56.67 (Me), 122.40 (vinylic), 141.83 (vinylic); IR (KBr) m (cmÀ1
):
3200, 3070, 2914, 2864, 2720, 1686, 1578, 1550, 1468, 1349,
970, 720, 580; MS (ESI): 270.1 ([M+H])+
.
(5a). (2E)-3-(3,5-dimethoxyphenyl)-1-(3-hydroxynaphthalen-2
-yl) prop-2-en-1-one. M.p 118–120 °C. 1
H NMR (500 MHz, CDCl3):
o 12.62 (s, 1H, OH), 7.92 (dd, 1H, J = 10.0, H-100
), 7.85 (d, 1H,
J = 10.5, Ha), 7.82 (dd, 1H, J = 10.5, H-50
), 7.69 (t, 1H, J = 9.5, H-70
),
7.58 (dd, 1H, J = 9.5, H-80
), 7.55 (m, 1H, J = 8.5, H-60
), 7.50 (d, 1H,
J = 9.0, H-30
), 7.09 (dd, 2H, H-2 & H-6), 7.07 (d, 1H, J = 10.5, Hb),
6.75 (s, 1H, H-4), 3.90 (s, 6H); 13
C NMR (125 MHz, DMSO-d6) o
198.5 (C@O), 118.1 (C10
), 167.2 (C20
), 124.2 (C30
), 140.4 (C40
),
135.3 (C50
), 135.2 (C60
), 124.9 (C70
), 130.2 (C80
), 124.1 (C90
), 134.5
(C100
), 144.0 (vinylic), 133.2 (vinylic), 138.2 (C1), 106.6 (C2 &
C6), 163.3 (C3 & C5), 106.2 (C4), 55.2 (Me); IR (KBr) m (cmÀ1
):
3062, 2947, 2729, 2514, 1692, 1597, 1512, 1436,1208, 1054, 871,
732; MS (ESI): 335.1 ([M+H])+
.
2.3. Method for the synthesis of hydroxy derivatives of
naphthylchalcones (1b–5b)
A solution of BBr3 (2.5 mL for each methoxy group) was added
to a cooled solution of the corresponding methoxy naphthylchal-
cone (1 mmol) in CH2Cl2 under argon. The cooling bath was
removed and the dark solution was warmed to RT and stirred for
1–5 h. The dark solution was then poured into ice water and fil-
tered. The aqueous layer was extracted with chloroform twice.
S. Radhakrishnan et al. / Bioorganic Chemistry 63 (2015) 116–122 117

3. The organic extract was washed with water followed by brine and
anhydrous sodium sulfate. This was then rotary evaporated to give
the respective product.
(1b). (2E)-3-(2,5-dihydroxyphenyl)-1-(3-hydroxynaphthalen-2
-yl) prop-2-en-1-one. M.p: 134–136 °C. 1
H NMR (500 MHz, CDCl3):
o 12.25 (s, 1H, OH), 10.60 (s, OH), 8.09 (dd, 1H, J = 12.5, H-100
), 7.87
(d, 1H, J = 11.5, Ha), 7.05 (s, 1H, H-6), 7.82 (dd, 1H, J = 10.5, H-50
),
7.67 (dd, 1H, J = 8.0, H-70
), 7.62 (dd, 1H, J = 9.5, H-80
), 7.57 (dd,
1H, J = 8.5, H-60
), 7.42 (d, 1H, J = 9.5, H-30
), 7.17 (t, 1H, J = 10.0,
H-4), 6.91 (dd, 1H, J = 9.0, H-3), 6.89 (d, 1H, J = 11.5, Hb), 5.70 (s,
OH); 13
C NMR (125 MHz, DMSO-d6) o 195.2 (C@O), 118.2 (C10
),
165.7 (C20
), 124.2 (C30
), 135.2 (C40
), 130.1 (C50
), 132.5 (C60
), 130.4
(C70
), 129.0 (C80
), 122.2 (C90
), 130.8 (C100
), 145.4 (vinylic), 130.5
(vinylic), 120.3 (C1), 155.7 (C2), 118.3 (C3), 125.6 (C4), 149.2
(C5), 118.2 (C6); IR (KBr) m (cmÀ1
): 3285, 3032, 2965, 2873, 2755,
1750, 1662,1627, 1561, 1431, 1377, 1210, 966, 719; HRMS m/z:
307.0963 ([M+H])+
; Calcd: 307.0970.
(2b). (2E)-3-(2,4-dihydroxyphenyl)-1-(3-hydroxynaphthalen-2
-yl) prop-2-en-1-one. M.p: 164–166 °C. 1
H NMR (500 MHz, CDCl3):
o 12.45 (s, 1H, OH), 10.54 (s, OH), 9.70 (s, OH), 8.15 (dd, 1H, J = 15.0,
H-100
), 7.92 (d, 1H, J = 12.0, Ha), 7.85 (dd, 1H, J = 10.0, H-50
), 7.78
(dd, 1H, J = 10.5, H-6), 7.62 (t, 1H, J = 9.5, H-70
), 7.55 (dd, 1H,
J = 8.0, H-80
), 7.51 (dd, 1H, J = 9.0, H-60
), 7.49 (d, 1H, J = 8.5, H-30
),
7.09 (d, 1H, J = 12.0, Hb), 6.59 (dd, 1H, J = 11.0, H-5), 6.47 (s, 1H,
H-3); 13
C NMR (125 MHz, DMSO-d6) o 197.8 (C@O), 117.5 (C10
),
167.0 (C20
), 122.4 (C30
), 139.1 (C40
), 130.4 (C50
), 134.5 (C60
), 125.5
(C70
), 129.2 (C80
), 124.2 (C90
), 135.2 (C100
), 142.4 (vinylic), 130.2
(vinylic), 120.2 (C1), 166.7 (C2), 118.3 (C3), 169.2 (C4), 105.2
(C5), 130.2 (C6); IR (KBr) m (cmÀ1
): 3129, 3045, 2657, 2781, 2614,
1683, 1614, 1500, 1437, 1335, 1248, 1166, 967, 662; HRMS m/z:
307.0965 ([M+H])+
; Calcd: 307.0970.
(3b). (2E)-1-(3-hydroxynaphthalen-2-yl)-3-(2-hydroxy-4-nitro
phenyl) prop-2-en-1-one. M.p: 153–155 °C.1
H NMR (500 MHz,
CDCl3): o 12.50 (s, 1H, OH), 10.55 (s, OH), 8.27 (s, 1H), 7.93 (d,
1H, J = 12.5, Hb), 7.89 (m, 1H, J = 10.5, H-6), 7.79 (d, IH, J = 12.5,
Ha), 7.77 (d, 1H, J = 9.5, H-60
), 7.70 (d, 1H, J = 7.5, H-5), 7.57 (dd,
1H, J = 8.5, H-40
), 7.09 (dd, 1H, J = 9.5, H-30
), 6.97 (m, 1H, J = 10.5,
H-50
); 13
C NMR (125 MHz, DMSO-d6) o 197.5(C@O), 130.4 (C60
),
120.3 (C10
), 118.2 (C50
), 138.2 (C40
), 119.4 (C30
), 164.9 (C20
), 128.2
(C1), 165.4(C2), 112.2 (C3), 137.5 (C4), 122.7 (C5), 118.2 (C6),
138.0 (vinylic), 140.7 (vinylic); IR (KBr) m (cmÀ1
): 3370, 3254,
3085, 2960, 2869, 1697, 1682, 1532, 1479, 1365, 984, 760, 687;
HRMS m/z: 336.0866 ([M+H])+
; Calcd: 336.0872.
(4b). (2E)-1-(3-hydroxynaphthalen-2-yl)-3-(4-hydroxy-3-nitro
phenyl) prop-2-en-1-one. Mp: 110–112 °C.1
H NMR (500 MHz,
CDCl3): o 12.45 (s, 1H, OH), 10.57 (s, OH), 8.15 (s, 1H), 7.89
(d,1H, J = 13.5, Hb), 7.77 (d, 1H, J = 10.0, H-60
), 7.74 (d, IH, J = 13.5,
Ha), 7.69 (d, 1H, J = 10.0, H-5), 7.55 (d, 1H, J = 7.5, H-6), 7.54 (t,
1H, J = 8.0, H-40
), 6.90 (dd, 1H, J = 9.0, H-30
), 6.90 (dd, 1H, J = 9.0,
H-50
); 13
C NMR (125 MHz, DMSO-d6) o 194.2(C@O), 130.5 (C60
),
120.5 (C10
), 118.7 (C50
), 136.7 (C40
), 117.8 (C30
), 160.2 (C20
), 125.9
(C1), 120.4 (C2), 139.2 (C3), 159.5 (C4), 112.4 (C5), 114.2
(C6),127.4 (vinylic), 147.3 (vinylic); IR (KBr) m (cmÀ1
): 3230,
3074, 2954, 2855, 1720, 1687, 1558, 1468, 1369, 1267, 1156,
920, 836, 660; HRMS m/z: 336.0864 ([M+H])+
; Calcd: 336.0872.
(5b). (2E)-3-(3,5-dihydroxyphenyl)-1-(3-hydroxynaphthalen-2
-yl)prop-2-en-1-one. M.p: 148–150 °C. 1
H NMR (500 MHz, CDCl3):
o 12.20 (s, 1H, OH), 10.75 (s, OH), 7.97 (dd, 1H, J = 10.0, H-100
), 7.84
(d, 1H, J = 11.5, Ha), 7.79 (dd, 1H, J = 9.5, H-50
), 7.73 (t, 1H, J = 9.0,
H-70
), 7.57 (dd, 1H, J = 7.5, H-80
), 7.54 (dd, 1H, J = 8.5, H-60
), 7.49
(d, 1H, J = 8.0, H-30
), 7.12 (dd, 2H, H-2 & H-6), 6.97 (d, 1H,
J = 11.5, Hb), 6.74 (s, 1H, H-4), 5.95 (s, OH); 13
C NMR (125 MHz,
DMSO-d6) o 200.2 (C@O), 120.1 (C10
), 168.2 (C20
), 125.3 (C30
),
139.7 (C40
), 136.2 (C50
), 137.2 (C60
), 125.2 (C70
), 132.6 (C80
), 127.1
(C90
), 135.2 (C100
), 144.7 (vinylic), 135.2 (vinylic), 137.1 (C1),
108.6 (C2 & C6), 160.3 (C3 & C5), 108.7 (C4); IR (KBr) m (cmÀ1
):
3278, 3041, 2955, 2870, 2760, 1749, 1659, 1549, 1424, 1376,
1224, 972, 724; HRMS m/z: 307.0967 ([M+H])+
; Calcd: 307.0970.
2.4. Enzyme activity assay
The mushroom tyrosinase inhibition activity of hydroxy naph-
thylchalcone compounds was measured using L-DOPA as substrate.
The activity assay used 3 ml of reaction medium containing
0.5 mM L-DOPA in 50 mM Na2HPO4–NaH2PO4 buffer (pH 6.8).
The final concentrations of tyrosinase was 3.33 mg/ml. The sub-
strate reaction progress curve was analyzed to obtain the reaction
rate constants. The reaction was carried out at 30 °C and pH 6.8.
2.5. Determination of the inhibition type of active compounds on
mushroom tyrosinase
Inhibitors were first dissolved in DMSO and used for the test
after a 30-fold dilution. The final concentration of DMSO in the test
solution was 3.33%. Mushroom tyrosinase (50 lL; 0.2 mg mLÀ1
)
was incubated with 50 lL of various concentrations of enzyme
substrate and 50 lL of phosphate buffer, and then 50 lL of
different concentrations of tested samples were simultaneously
added to the reaction mixtures. The measurement was performed
in triplicate for each concentration and averaged before further
calculation. The absorbance variations from these studies were
used to generate Lineweaver–Burk plots to determine the inhibi-
tion type. The kinetic parameter (Km) of the tyrosinase activity
was calculated by linear regression from Lineweaver–Burk plots.
For the type of enzyme inhibition and the inhibition constant (Ki)
for an enzyme-inhibitor complex, the mechanisms were analyzed
by Dixon plot, which is a graphical method plot of 1/enzyme
velocity (1/V) versus inhibitor concentration (I) with varying
concentrations of the substrate.
2.6. In silico docking between tyrosinase and target compounds
To further understand the binding modes of the hydroxy
naphthylchalcone compounds with mushroom tyrosinase,
molecular docking studies of compounds 1b–5b were performed
using Discovery Studio 4.5 (Accelrys, San Diego, CA, USA). To model
the tyrosinase structure, we used the crystal structure of
Agaricus bisporus tyrosinase (PDB ID: 2Y9X), A chain. From the
docking results, we checked for possible hydrogen-bonding and
non-bonding interactions with the amino acid residues.
3. Results and discussion
3.1. Chemistry
The parent naphthylchalcones were synthesized by the base
catalyzed Claisen–Schmidt condensation of 20
-hydroxy-10
-aceto
napthone with an appropriate aldehyde in a polar solvent like
methanol. The methoxy naphthylchalcones were then successfully
dealkylated to their corresponding hydroxy compounds in the
presence of boron tribromide (Scheme 1).
The structures of the compounds synthesized were confirmed
by 1
H NMR, 13
C NMR, FTIR and HRMS. All the hydroxy naph-
thylchalcone compounds (1b–5b) exhibited better tyrosinase inhi-
bitions in comparison with the reference standard, kojic acid
(Table 1).
In terms of the structure–activity relationships, compound 5b
[(2E)-3-(3,5-dihydroxyphenyl)-1-(3-hydroxynaphthalen-2-yl)prop-
2-en-1-one] exhibited the most potent tyrosinase inhibitory activi-
ties with inhibitions of 68.5%. The hydroxyl groups in compounds
carry out the nucleophilic attack on the copper atoms in the active
118 S. Radhakrishnan et al. / Bioorganic Chemistry 63 (2015) 116–122

4. site of the enzyme and are directly involved in transferring protons
during catalysis, which then result in inactivation of the tyrosinase.
Moreover, the tyrosinase inhibitory activity was higher when the
hydroxyl group was introduced in the p-position with respect to
the o-position. Compound 2b (2E)-3-(2,4-dihydroxyphenyl)-1-(3-
hydroxynaphthalen-2-yl) prop-2-en-1-one with a 2,4-substituted
resorcinol structure showed threefold better tyrosinase inhibition
(Ki: 4.5 lM) than kojic acid (Ki: 12.2 lM), probably because
it results in a molecular skeleton closely similar to that of
L-tyrosine. Compound 1b with a 2,5-dihydroxy substitution showed
moderate (56.2%) tyrosinase inhibition.
Further, we have determined the effect of electron withdrawing
substituents on ring B. The introduction of electron-withdrawing
groups on ring B may increase the electrophilicity of the b-carbon,
thus improving the bioactivity of the resulting compounds.
Presence of a strong electron releasing group in the ortho position
(AOH) and an electron withdrawing group (ANO2) in the para
position in compound 3b modulates the electronic structure of ring
B significantly, that could be accounted to its better tyrosinase
inhibitory potential (55.5%). However, substitution with less
activating para hydroxy substituent resulted in a fall of tyrosinase
inhibitory activity as seen with compound 4b (51.2%). Results
indicated that electron-donating groups contributed more to the
inhibitory activity of the compounds on mushroom tyrosinase
than the electron-withdrawing groups. Electron donating groups
(e.g. AOMe, AOH) on the atoms adjacent to the p system activate
the aromatic ring by increasing the electron density on the ring
through a resonance donating effect.
3.2. Kinetics
In the present study, tyrosinase extracted from the edible
mushroom A. bisporus is used due to its easy availability and high
homology with the mammalian enzyme that renders it well suited
as a model for studies on melanogenesis [23]. We used L-DOPA as
substrate for the effect of inhibitor compounds (diphenolase activ-
ity) on the oxidation of L-DOPA by tyrosinase [8]. Results indicated
that both compounds 5b and 2b could inhibit the diphenolase
activity of tyrosinase in a dose-dependent manner. With increasing
concentrations of inhibitors, the remaining enzyme activity
Scheme 1. (General method for synthesis of hydroxy naphthylchalcones 1b–5b). Reagents and conditions: (a) MeOH, NaOH, 0 °C, 24 h; (b) BBr3, CH2Cl2.
Table 1
Inhibition effects and docking results of hydroxy naphthylchalcones (1b–5b) on polyphenol oxidase.
Compound Tyrosinase inhibition at 50 lM*
(%) CDOCKER energy (kcal/mol) Type of interactions Donor–acceptor Distance (Å)
1b 56.2 ± 0.12 À22.35 Intramolecular H-bonding 20
-OHÁ Á ÁO@C 2.02
Metal coordination bond Cu401Á Á ÁOH-5 2.53
Hydrophobic p–p stacking Ring BÁ Á ÁHis263 3.55
2b 62.5 ± 0.45 À19.25 H-bonding 2-OHÁ Á ÁOd1 (Asn260) 1.99
Hydrophobic p–p stacking Ring BÁ Á ÁHis263 4.07
Hydrophobic p–r Ring BÁ Á ÁVal283 2.75
3b 55.5 ± 0.25 À16.83 Intramolecular H-bonding 20
-OHÁ Á ÁO@C 1.97
Metal coordination bond Cu401Á Á ÁO2N 2.40
H-bonding 2-OHÁ Á ÁOd1 (Asn260) 1.98
Hydrophobic p–r Ring BÁ Á ÁVal283 2.56
Hydrophobic p–p stacking Ring BÁ Á ÁHis263 4.40
4b 51.2 ± 0.68 À16.23 H-bonding Asn260 Hd22Á Á ÁO@C 2.59
H-bonding 4-OHÁ Á ÁO@C (Met280) 2.80
H-bonding (His85) Hd2Á Á ÁO2N 2.52
H-bonding (His259) HƐ1Á Á ÁO2N 2.46
p-cationic bonding (Arg268) NH1Á Á Áring A 4.02
Hydrophobic p–p stacking Ring BÁ Á ÁHis263 4.32
5b 68.5 ± 0.22 À22.36 H-bonding 3-OHÁ Á ÁO@C (Met280) 1.91
Metal coordination bond Cu401Á Á ÁOH-5 1.95
Hydrophobic p–p stacking Ring BÁ Á ÁHis263 3.64
Kojic acid 49.5 ± 0.66 À11.69 H-bonding Asn260 HaÁ Á ÁO 2.93
H-bonding Val283 HaÁ Á ÁO 2.64
H-bonding HÁ Á ÁO@C (Met280) 2.84
*
Naphthylchalcone derivatives were synthesized according to the details in Scheme 1; Values indicate means ± SE for three determinations.
S. Radhakrishnan et al. / Bioorganic Chemistry 63 (2015) 116–122 119

5. decreased exponentially. The inhibitor concentration leading to
50% activity lost (IC50) for compounds 5b and 2b was estimated
to be 10.4 lM and 14.4 lM, respectively (Fig. 1).
The plots of the remaining enzyme activity versus the
concentration of enzyme at different inhibitor concentrations gave
a family of straight lines, which all passed through the origin. The
presence of inhibitor did not reduce the amount of enzyme, but
just resulted in the inhibition of enzyme activity. The results
showed that both the compounds 5b and 2b were reversible inhi-
bitors of mushroom tyrosinase for oxidation of L-DOPA (Fig. 2).
In this study, we investigated in depth the tyrosinase inhibitory
activities of compounds 5b and 2b. We measured the reaction rates
in the presence of active inhibitors with various concentrations of
L-DOPA as a substrate. As the concentrations of active inhibitors 5b
and 2b increased, Km values gradually increased, but Vmax values
did not change, thereby indicating the inhibitors act as competitive
inhibitors of mushroom tyrosinase (Fig. 3).
Data were obtained as mean values of 1/V, the inverse of the
absorbance increase at a wavelength of 492 nm per min of three
independent tests with different concentrations of L-DOPA as a
substrate. The concentration of compounds 5b and 2b from top
to bottom is 20 lM, 5 lM, 1.25 lM and 0 lM, respectively. The
inhibition kinetics were illustrated by Dixon plots, which were
obtained by plotting 1/V versus [I] with varying concentrations of
substrate. Dixon plots gave a family of straight lines passing
through the same point at the second quadrant, giving the inhibi-
tion constant (Ki) (Fig. 4).
The Ki value estimated from this Dixon plot was 3.8 lM and
4.5 lM for the compounds 5b and 2b, respectively. A comparison
of the Ki values of the compounds with that of kojic acid revealed
that they possess much higher affinity to tyrosinase than kojic acid
(Table 2).
3.3. Docking studies
We simulated binding between the active site of mushroom
tyrosinase and the active inhibitors 5b and 2b using Accelrys Dis-
covery Studio 4.5 suite.). Fig. 5 shows selected docked conforma-
tions of compounds 5b and 2b along with the positive control,
kojic acid in the tyrosinase binding site (see Fig. 5).
Additionally, we searched for hydrogen bonding interactions
between mushroom tyrosinase and the inhibitor compounds or
kojic acid. Docking results show that compound 5b (À22.36
kcal molÀ1
) combines with mushroom tyrosinase more strongly
than compound 2b (À19.25 kcal molÀ1
) (Table 1). Copper ion
(Cu401) was strongly bound by the hydroxyl group (3-OH) of com-
pound 5b at a distance of 1.95 Å. Tyrosinase substrates like L-DOPA
20
40
60
80
100
120
RelaƟveacƟvity(%)
0
0 2 4 6 8 10 12
[I] (μM)
14 16 18
5b
2b
Fig. 1. Inhibition effects of compounds 5b and 2b on the diphenolase activity of
mushroom tyrosinase. Data are presented as means (n = 3).
0
50
100
150
200
250
0 2 4 6 8 10 12
Activity(µM/min)
[E] ( µg/mL)
0
1
2
3
4
0
20
40
60
80
100
120
140
160
180
0 2 4 6 8 10 12
Activity(µM/min)
[E] (µg/mL)
4
1
2
3
0
5b
2b
Fig. 2. The inhibitory mechanism of compounds 5b and 2b on mushroom
tyrosinase were reversible. The concentration of inhibitor used for curves 0–4 are
0, 0.25, 0.5, 1.0 and 2.0 lM respectively.
-200
-100
0
100
200
300
400
-4 -2 0 2 4 6
1/V(µM/min)-1
1/S (mM/L)
0 mM
0.05 mM
0.10 mM
0.15 mM
-200
-100
0
100
200
300
400
-4 -2 0 2 4 6
1/V(µM/min)-1
1/S (mM/L)
0 mM
.05 mM
0.10 mM
0.15 mM
5b
2b
Fig. 3. Lineweaver Burk plot for inhibition of compounds 5b and 2b on mushroom
tyrosinase. Data were obtained as mean values of 1/V, the inverse of the absorbance
increase at a wavelength of 492 nm per min of three independent tests with
different concentrations of L-DOPA as a substrate. The concentration of compounds
5b and 2b from top to bottom is 20 lM, 5 lM, 1.25 lM and 0 lM respectively.
120 S. Radhakrishnan et al. / Bioorganic Chemistry 63 (2015) 116–122

6. also bind to the copper ion of tyrosinase via their OH group. The
5-OH group of compound 5b formed a strong hydrogen bond
(1.91 Å) with the carboxylate oxygen of Met280. Compound 1b
with an IC50 of 20.4 lM showed coordination with Cu401 at a dis-
tance of 2.53 Å. However, the formation of a strong intramolecular
hydrogen bond (2.02 Å) between the 20
-OH and the carbonyl group
may hamper the latter to interact with the receptor. The ligand 2b
(IC50: 14.4 lM) does not interact with the bi-nuclear copper-
binding site, but mainly with residues’ side chains in the active-
site entrance. The interaction of OH-2 of ring B in compound 2b
with Asn260 (1.99 Å) emphasizes the importance of this residue
located close to the entrance of the receptor. The catalytic pocket
of the enzyme is hydrophobic. Ring B showed hydrophobic p–p
stacking interactions with His263 while p–r interactions were
seen with Val283. For kojic acid, it was observed that the com-
pound forms hydrogen bonds with Asn260, Met280, Val283 and
also a p–p interaction with His283. This indicates that the hydroxy
derivatives of naphthylchalcones might inhibit tyrosinase activity
by binding at the active site of mushroom tyrosinase.
4. Conclusion
To summarize, in this paper, we synthesized novel hydroxy
derivatives of naphthylchalcones, and studied their inhibition on
-40
-30
-20
-10
0
10
20
30
40
50
-40 -30 -20 -10 0 10 20 30
1/v(µM/min)-1
[inhibitor] µM
2b
-40
-30
-20
-10
0
10
20
30
40
50
-40 -30 -20 -10 0 10 20 30
1/v(μM/min)-1
[inhibitor] μM
5b
Fig. 4. Dixon plot for the inhibitory effect of compounds 5b and 2b on L-DOPA
oxidation catalyzed by mushroom tyrosinase. The inhibitor concentrations were 0,
10 lM and 20 lM respectively. The L-DOPA concentrations were 200, 400 and
600 lM.
Table 2
Effect on mushroom polyphenol oxidase activity and kinetic analysis of compounds.
Compound Type of inhibition$
IC50
*
(lM) Ki
#
(lM)
1b Competitive 20.4 ± 0.25 10.8
2b Competitive 14.4 ± 0.44 4.5
3b Competitive 22.9 ± 0.14 11.2
4b Competitive 25.6 ± 0.11 11.9
5b Competitive 10.4 ± 0.65 3.8
Kojic acid – 27.5 ± 0.56 12.2
*
(IC50): refers to the concentration of compound that caused 50% inhibition.
#
Values were measured at 5 lM of active compounds and Ki is the (inhibitor
constant).
$
Lineweaver–Burk plot of mushroom tyrosinase: Data are presented as mean
values of 1/V, which is the inverse of the increase in absorbance at wavelength
492 nm/min (DA492/min), for three independent tests with different concentra-
tions of L-DOPA as the substrate.
Kojic
acid
2b
5b
Fig. 5. Docking result of compounds 5b, 2b & kojic acid in the tyrosinase catalytic
pocket. Ligands are displayed as ball and stick while the core amino acid residues
are displayed as stick. The green dotted lines show the hydrogen bond interactions
and the purple lines show the non-bonding interactions. The ochre balls represent
the copper ions. (For interpretation of the references to color in this figure legend,
the reader is referred to the web version of this article.)
S. Radhakrishnan et al. / Bioorganic Chemistry 63 (2015) 116–122 121

7. the diphenolase activity of mushroom tyrosinase. Compounds 5b
and 2b were found to be significantly more potent than kojic acid
with their IC50 values of 10.4 lM and 14.4 lM respectively. SAR
studies have identified hydroxyl as the functional group indispens-
able for tyrosinase inhibitory activity. Another structural advan-
tage could be attributed to the naphthyl ring that could exert a
nucleophilic action on the dihydroxy phenyl ring. Both the com-
pounds exhibited reversible competitive inhibition. Compound
2b with a 2,4-substituted resorcinol structure in the B-ring had
structural resemblance to the substrate L-tyrosine. Moreover, the
binding of the inhibitor via a coordinate bond will ensure that
access to the active site by the substrate is effectively blocked. This
could curb the enzymes ability to oxidize the substrates subse-
quently leading to an inhibition in mushroom tyrosinase. These
preliminary findings justify pursuing further work on hydroxy sub-
stituted naphthylchalcones to serve as a potential scaffold for the
future development of active tyrosinase inhibitors. New synthetic
efforts will be made to obtain structural analogues by introducing
a variety of substituents at the two aromatic rings to help refine
the requirements for optimal activity.
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